Methods of inhibiting chemokine binding to chemokine receptors

ABSTRACT

Novel peptides or peptidomimetic agents or small molecules for modulating the biological effect of a chemokine. According to the present invention, the therapeutic agents preferably are endowed with the capacity to bind to certain chemokines in order to modulate the biological interaction between the target ligand, chemokine, and the respective target receptor, chemokine receptor. These peptides may be described as agonist ligands or antagonists. Next, preferably certain peptides share consensus sequences are described which characterize the families or categories of these modulator peptides.

This Application is a Continuation-in-Part of PCT Application No.PCT/IL03/00155, filed 27 Feb. 2003, currently pending, which claimspriority from 60/359,995, filed 28 Feb. 2002, now expired, all of whichare hereby incorporated by reference as if fully set forth herein.

FIELD OF THE INVENTION

The present invention discloses novel peptidic molecules orpeptidomimetic agents, which are capable of binding chemokines andmodulating their biological functions.

BACKGROUND OF THE INVENTION

Drug discovery in the post-genomics era provides enormous opportunitiesas well as new challenges. The targets of the drug discovery processhave changed greatly over the last 50 years. The development of advancedpurification technologies and the tools of molecular biology havebrought molecular targets into the current discovery process. In thelast ten years, there has been a trend towards selecting moleculartargets for the screening process, and the human and other genomeprojects have made available many thousands of additional targets fordrug discovery.

In addition to these novel targets with unknown potential, there are asignificant number of well-validated targets associated with major humandiseases. Most of these are either nuclear receptors or Gprotein-coupled receptors. It was found, that in some cases, onecompound that has an effect through one receptor, can also act throughanother receptor, and that several compounds can work through the samereceptor. Unfortunately, even when the mechanism of a disease process isunderstood, for example by identifying the receptor(s) responsible forsuch a process, this information has not always resulted in thedevelopment of new treatments. For example, subjects who suffer frominflammation associated diseases and disorders have a great anddesperate demand for novel drugs as therapeutic agents. Currenttherapies are merely palliative and have not been significantly improvedin recent years.

Recent scientific advances provide some hope that new treatments willsoon be available for these diseases. Sequencing of the human genome,which contains nearly 30,000 genes, has been recently completed. Thissignificant achievement in the frontiers of human medicine will allowthe identification of genes involved in the onset and progression ofhuman diseases and pathological states. Many of these genes will serveas valid targets in the discovery process of drugs that are moreeffective in the treatment of inflammatory diseases. Along with amassive flow of novel genes with potential therapeutic properties, thereis a growing need for more rapid and efficient ways to discover leadcompounds with enhanced (agonistic) or inhibitory (antagonistic)properties.

Chemokines are among the biological factors that are, amongst otherfunctions, involved in the inflammatory disease process. Chemokinesbelong to a group of small, ˜8-14 kDa, mostly basic, heparin bindingproteins that are related both in their primary structure and thepresence of 4 conserved cysteine residues. The chemokines arechemotactic cytokines that have been shown to be selectivechemoattractants for leukocyte sub-populations in vitro, and to elicitthe accumulation of inflammatory cells in vivo. In addition tochemotaxis, chemokines mediate leukocyte de-granulation (Baggiolini andDahinden, 1994) and the up-regulation of adhesion receptors (Vaddi andNewton, 1994), and have recently been implicated in the suppression ofhuman immunodeficiency virus replication (Cocchi et al., 1995).

Chemokines can be divided into 4 groups (CXC, CX3C, CC, and C) accordingto the positioning of the first 2 closely paired and highly conservedcysteines of the amino acid sequence. The specific effects of chemokineson their target cells, are mediated by members of a family of7-transmembrane-spanning G-protein-coupled receptors. These chemokinereceptors are part of a much bigger super family of G-protein-coupledreceptors that include receptors for hormones, neurotransmitters,paracrine substances, inflammatory mediators, certain proteinases, tasteand odorant molecules and even photons and calcium ions.

The chemokine receptors have received increasing attention due to theircritical role in the progression of immune disease states such asasthma, atherosclerosis, graft rejection, AIDS, multiple sclerosis andothers. It would be useful to have therapeutic agents capable ofinhibiting the binding of ligands of some chemokine receptors in orderto lessen the intensity of or cure these diseases. Chemokines themselvesplay an essential role in the recruitment and activation of cells fromthe immune system. They also have a wide range of effects in manydifferent cell types beyond the immune system, including for example, invarious cells of the central nervous system (Ma et al., 1998) orendothelial cells, where they result in either angiogenic or angiostaticeffects (Strieter et al., 1995). Recent work has shown that particularchemokines may have multiple effects on tumors, including promotinggrowth, angiogenesis, metastasis, and suppression of the immune responseto cancer, while other chemokines inhibit tumor mediated angiogenesisand promote anti-tumor immune responses.

Recently, it was shown that the SDF-1α/CXCR4 chemokine/chemokinereceptor pathway is involved in dissemination of metastatic breastcarcinomas (Muller A, 2001). This example illustrates that bothchemokines and their receptors are potentially valuable targets fortherapeutic intervention in a wide range of diseases.

SUMMARY OF THE INVENTION

The background art does not teach or suggest sequences or compositionscontaining peptidic modulators capable of binding to chemokines andinhibiting or activating their biological functions. The background artalso does not teach or suggest sequences or compositions containing thebasic consensus sequences that characterize families of such peptidicchemokine-binding modulators. In addition, the background art does notteach or suggest the nucleic acid molecules encoding for such peptidicchemokine-binding modulators. Finally, the background art does not teachor suggest methods of treatment employing such peptidicchemokine-binding modulators.

The present invention overcomes these deficiencies of the background artby providing peptidic chemokine-binding modulators, with defined aminoacid sequences, which have been found to bind to specific chemokines,including but not limited to human SDF-1α, MIG, IL-8, MCP-1 and Eotaxin,and which modulate the binding of these chemokines to their respectivereceptors and/or which otherwise have an inhibitory or stimulatoryeffect on the biological activity of chemokines.

It should be noted that the term “peptidic” as used herein, alsoincludes peptidomimetics and hybrid structures comprised of at least oneamino acid and at least one molecule that is not an amino acid, or atleast not a naturally occurring amino acid. The term amino acid mayoptionally also include non-naturally occurring amino acids as well asnaturally occurring amino acids, and derivatives and analogs thereof.

A peptide mimetic (peptidomimetic) is a molecule that mimics thebiological activity of a peptide, yet is no longer peptidic in chemicalnature. By strict definition, a peptidomimetic is a molecule that nolonger contains any peptide bonds, i.e., amide bonds between aminoacids; however, in the context of the present invention, the termpeptide mimetic and also the term peptidomimetic are intended to includemolecules that are no longer completely peptidic in nature, such aspseudo-peptides, semi-peptides and peptoids. Whether completely orpartially nonpeptide, peptidomimetics according to the present inventionprovide a spatial arrangement of reactive chemical moieties that closelyresembles the three-dimensional arrangement of active groups in thepeptide on which the peptidomimetic is based. The techniques ofdeveloping peptidomimetics are conventional. Thus, non-peptide bondsthat allow the peptidomimetic to adopt a similar structure to theoriginal peptide can replace peptide bonds.

Replacing chemical groups of the amino acids with other chemical groupsof similar structure can also be used to develop peptidomimetics.

Preferably, the present invention features basic consensus sequences,some of which characterize families of such peptidic chemokine-bindingmodulators.

More specifically, if the biological activity caused by the targetprotein, i.e. chemokine receptor, when activated through binding thetarget ligand, i.e. chemokine, involves activation of some biologicalfunction, then the inhibitory peptide preferably inhibits suchactivation. On the other hand, if the biological activity caused by thereceptor involves inhibition of some biological function, then theinhibitory peptide preferably blocks inhibition of the biologicalfunction.

According to another preferred embodiment of the present invention,there is provided a composition for treating inflammatory and cancermetastasis conditions in a subject, comprising a pharmaceuticallyeffective amount of a therapeutic agent for administering to thesubject, the therapeutic agent being composed of the peptidicchemokine-binding modulators as the active ingredient as well as asuitable pharmaceutical carrier if necessary.

According to a preferred embodiment of the present invention, thetherapeutic agent may be administered topically, intranasally and byinhalation. Alternately, the therapeutic agent may be administered bysystemic administration.

According to the present invention, there is provided a peptidicchemokine modulator for modulating a biological effect of a chemokine,comprising a molecule having a defined amino acid composition.

According to another embodiment of the present invention, there isprovided a peptidic chemokine modulator for modulating a biologicaleffect of a chemokine, comprising a molecule composed of the amino acidsH, S, A, L, I, K, R, T and P, and featuring at least 2 Histidines spreadalong the molecule, wherein the molecule features an overall positivecharge (family 1). The present invention comprises peptides having thesecharacteristics and preferably being up to about 20 amino acids inlength, more preferably from about 10 to about 20 amino acids in length,and optionally and most preferably about 12 amino acids in length.Preferably, the molecule comprises a peptide having an amino acidsequence selected from the group consisting of SIFAHQTPTHKN (seq idno:100), SIPSHSIHSAKA (seq id no:101), SAISDHRAHRSH (seq id no:96),SAGHIHEAHRPL (seq id no:95), ACHASLKHRC (seq id no. 44), AHSLKSITNHGL(seq id no:46), ESDLTHALHWLG (seq id no:54), HSACHASLKHRC (seq idno:69), WSAHIVPYSHKP (seq id no:143), YATQHNWRLKHE (seq id no:145),CAHLSPHKC (seq id no:1), GVHKHFYSRWLG (seq id no:61), HPTTPFIHMPNF (seqid no:66), SVQTRPLFHSHF (seq id no:113), and VHTSLLQKBPLP (seq idno:133). More preferably, the peptide has an amino acid sequenceSIFAHQTPTHKN (seq id no:100). The peptidic chemokine modulator mayoptionally be used for binding to a chemokine selected from the groupcomprising MIG, MCP-1, IL-8, SDF-1 alpha and Eotaxin.

According to another embodiment of the present invention, there isprovided a peptidic chemokine modulator for modulating a biologicaleffect of a chemokine, comprising a molecule composed of the amino acidsH, P, T, L, R, W, F, and featuring at least two neighboring histidines,wherein the molecule features an overall positive charge (family 2). Thepresent invention comprises peptides having these characteristics andpreferably being up to about 20 amino acids in length, more preferablyfrom about 10 to about 20 amino acids in length, and optionally and mostpreferably about 12 amino acids in length. Preferably, the moleculecomprises a peptide having an amino acid sequence selected from thegroup consisting of GDFNSGHHTTTR (seq id no:59), HHFHLPKLRPPV (seq idno:64), HHTWDTRIWQAF (seq id no:65), LDYPIPQTVLHH (seq id no:76),LLADTTHHRPWP (seq id no:79), TRLVPSRYYHHP (seq id no:125), CHHNLSWEC(seq id no:7) and SFWHHHSPRSPL (seq id no:99). More preferably, thepeptide has an amino acid sequence LLADTTHHRPWP (seq id no 79:).

According to another embodiment of the present invention, there isprovided at least one peptide having at least 90% sequence homology,more preferably about 95% sequence homology, and most preferablysequence identity to any peptide listed in any of the Tables hereinand/or in the specification, that is preferably up to about 20 aminoacids in length, more preferably from about 10 to about 20 amino acidsin length, and optionally and most preferably about 12 amino acids inlength. A peptide according to the present invention may optionallybelong to one of the two families, or may alternatively not belong toeither family.

Hereinafter, homology is defined as that which is determined with theSmith-Waterman algorithm, using the Bioaccelerator platform developed byCompugene (gapop: 10.0, gapext: 0.5, matrix: blosum62).

According to another embodiment of the present invention, there isprovided a composition for treating a condition involving abnormal cellmigration in a subject, the composition comprising a pharmaceuticallyeffective amount of a therapeutic agent for administering to thesubject, the therapeutic agent comprising a chemokine modulator asdescribed above. Preferably, the condition comprises an inflammatorycondition. Alternatively, the condition comprises cancer metastasis.Optionally, the therapeutic agent is administered by topicaladministration, such that the composition further comprises apharmaceutically acceptable carrier for topical administration.Preferably, the topical administration is to the skin of the subject.Optionally, the therapeutic agent is administered by inhalation, suchthat the composition further comprises a pharmaceutically acceptablecarrier for inhalation. Alternatively, the therapeutic agent isadministered intranasally, such that the composition further comprises apharmaceutically acceptable carrier for intranasal administration.

Optionally, the therapeutic agent is characterized by an ability toinhibit binding of the chemokine to a chemokine receptor.

Optionally, the therapeutic agent is characterized by an ability toenhance binding of the chemokine to a chemokine receptor.

According to another embodiment of the present invention, there isprovided a method for treating a disease modulated through and/or causedby binding of a chemokine to a chemokine receptor in a subject,comprising administering a pharmaceutically effective amount of atherapeutic agent to the subject, the therapeutic agent comprising apeptidic chemokine modulator as described above.

Preferably, the therapeutic agent binds to at least one of thechemokines and wherein the therapeutic agent directly modulates theactivity of the chemokine by modulation of binding to the chemokinereceptor.

Optionally and preferably, the disease is selected from the groupconsisting of: inflammation (primary or secondary), allergy, anon-optimal immune response, an autoimmune reaction (includingrheumatoid arthritis, systemic lupus erythematosis, multiple sclerosisand others), allograft rejection, diabetes, sepsis, cancer and any typeof malignant cell growth, acute and chronic bacterial and viralinfections, arthritis, colitis, psoriasis, atherosclerosis, hypertensionand reperfusion ischemia.

According to still another embodiment of the present invention, there isprovided an antibody for binding to a chemokine-binding receptor,comprising: an antibody being capable of recognizing at least a portionof a chemokine-binding receptor, wherein the antibody also recognizes apeptide having a sequence as described above.

Optionally, there is also provided a vaccine formed with the aboveantibody.

According to still another embodiment of the present invention, there isprovided a method for producing an antibody, comprising: inducingformation of antibody against a peptide having a sequence according tothe above description, wherein the antibody is also capable ofrecognizing a chemokine-binding receptor.

Preferably, the antibody comprises a monoclonal antibody. Alternatively,the antibody comprises a polyclonal antibody. Preferably, the antibodyforms a vaccine.

The peptidic chemokine-binding modulators are then preferably used todevelop one or more lead compounds for new therapies. Alternatively oradditionally, the peptidic chemokine-binding modulators themselves mayhave therapeutic value, and as such, may optionally be used fortreatment of a subject.

Also additionally or alternatively, binding of the peptidicchemokine-binding modulators may optionally be used to identify leadproteins, which are reference proteins whose reactivity descriptors aresubstantially similar to those of the protein of interest such as novelchemokine binding proteins. By “reactivity descriptor” it is meant thecharacteristics of binding to other chemokines and/or chemokine bindingproteins, and/or the biological activity induced and/or inhibited by thereference protein.

Also additionally or alternatively, the peptidic chemokine-bindingmodulators may optionally be used as antigens to produce antibodies tothese peptides, which can also bind chemokine-binding receptors, oroptionally may be used to stimulate the production of auto-antibodiesagainst these peptides that can also bind chemokine receptors. Thelatter use would involve using these peptides (or other modulators) as avaccine, optionally with any suitable vaccine carrier that could easilybe selected by one of ordinary skill in the art, including but notlimited to, adjuvants, carriers and the like. More preferably, themodulators that are used to produce the antibodies are selected from oneof the peptides described herein. It should be noted that the vaccinemay also optionally comprise the antibody itself.

Hereinafter, the term “biologically active” refers to molecules, orcomplexes thereof, which are capable of exerting an effect in abiological system. Hereinafter, the term “fragment” refers to a portionof a molecule or a complex thereof, in which the portion includessubstantially less than the entirety of the molecule or the complexthereof.

Hereinafter, the term “amino acid” refers to both natural and syntheticmolecules that are capable of forming a peptide bond with another suchmolecule. Hereinafter, the term “natural amino acid” refers to allnaturally occurring amino acids, including both regular and non-regularnatural amino acids. Hereinafter, the term “regular natural amino acid”refers to those alpha amino acids that are normally used as componentsof a protein. Hereinafter, the term “non-regular natural amino acid”refers to naturally occurring amino acids, produced by mammalian ornon-mammalian eukaryotes, or by prokaryotes, which are not usually usedas a component of a protein by eukaryotes or prokaryotes. Hereinafter,the term “synthetic amino acid” refers to all molecules which areartificially produced and which do not occur naturally in eukaryotes orprokaryotes, but which fulfill the required characteristics of an aminoacid as defined above. Hereinafter, the term “peptide” includes both achain and a sequence of amino acids, whether natural, synthetic orrecombinant. Hereinafter, the term “peptidomimetic” includes bothpeptide analogues and mimetics having substantially similar or identicalfunctionality thereof, including analogues having synthetic and naturalamino acids, wherein the peptide bonds may be replaced by other covalentlinkages.

BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES

The invention is herein described, by way of example only, withreference to the accompanying drawings and tables, wherein:

FIG. 1 shows the results of binding of chemokines to BKT-P1, BKT-P3,BKT-P22, and BKT-P37 peptides, which bind, respectively, to at least oneof the following chemokines, SDF-1α, MCP-1, Eotaxin and IL-8;

FIG. 2 shows the binding of synthetic peptides from Table 1 which caneither bind specifically to various chemokines (such as BKT-P 1, shownin FIG. 2B), or can alternatively bind specifically to a singlechemokine (such as BKT-P9 alone, as shown in FIG. 2A);

FIG. 3 shows the results of binding of one of the synthetic peptidesfrom family 1, BKT P3, to various chemokines. BKT-P18, which does notbelong to the family, is shown as a control;

FIG. 4 shows a graphical representation of the biological activityresults for BKT-P3, from family 1 in a biovalidation assay system inwhich the chemokine MIG and the adhesion receptor VCAM-1 are used toactivate the binding of T cells (see “Material and Methods”) (FIG. 4A);BKT-P10, which does not belong to the family, is shown as a control(FIG. 4B).

FIG. 5 shows a graphical representation of the level of immnunity (O.D.)of antibody raised against BKT-P3 and BKT-P2, both of which belong tofamily 1, to the relevant peptides, BKT-P3 and BKT-P2. The preimmuneserum of the same mice used as a control. “Blank” is the O.D. level ofantibody binding to the plastic (of the Elisa experiment), with noaddition of peptides.

Table 1 shows the sequences of peptides that bind to the chemokinesMCP-1, SDF-1α, MIG, Eotaxin and IL-8;

Table 2 shows a family of peptides (family no. 1) that bind to MIG,MCP-1, IL-8, SDF-1α and Eotaxin and are predominantly composed of theamino acids H, S, A, L, I, K, R, T and P, featuring at least 2Histidines spread along the molecule. The abundance of positivelycharged amino acids such as H, K and R, resulted in peptides having anoverall positive charge. The remaining amino acids, mentioned above,might participate in the determination of the three dimensionalstructure of the peptides;

Table 3 shows a family of peptides (family no. 2) that bind mostly toMCP-1 and in individual cases to IL-8, SDF-1α and Eotaxin; the bindingmotif for peptides in this family is predominantly composed of the aminoacids H, P, T, L, R, W, F, featuring at least 2 Histidines next to eachother. The abundance of positively charged amino acids, such as H and R,resulted in peptides having an overall positive charge. The remainingamino acids, mentioned above, might participate in the determination ofthe three dimensional structure of the peptides;

Table 4 shows a summary of the results of the biological activity offour representatives from family 1, in a biovalidation assay system, inwhich various chemokines and the adhesion receptor VCAM-1 are used toactivate the binding of T cells (as described in “Materials andMethods”).

Table 5 shows a summary of the results of the biological activity of tworepresentatives from Table 1, which do not belong to either family 1 orfamily 2, in a biovalidation assay system, in which various chemokinesand the adhesion receptor VCAM-1 are used to activate the binding of Tcells (as described in “Materials and Methods”).

Table 6 shows the list of synthetic peptides that were ordered forfurther analysis of their ability to modulate chemokine activity.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is composed of peptidic modulatory molecules withdefined amino acid sequences which have been found to bind to specificchemokines, including, but not limited to, human SDF-1α, MIG, IL-8,MCP-1 and Eotaxin, and which inhibit or stimulate the binding of thesechemokines to their respective receptor/s and/or which otherwise have aninhibitory or stimulatory effect on the biological activity ofchemokines. Preferably, the present invention specifies basic consensussequences, with overall positive electrostatic charge, whichcharacterize families of such modulatory chemokine-binding peptidemolecule.

The chemokine binding peptides mimic chemokine receptor structures ormotifs. It is therefore predicted that the chemokine-binding peptides,when injected or otherwise introduced into mice or humans, may produceantibodies against chemokine receptors that can be either used for theproduction of monoclonal antibodies or used for development of a vaccineagainst a particular chemokine receptor/s. Whether antibodies areproduced, or alternatively, binding to a particular chemokine receptoris blocked and/or enhanced, may depend upon the method of introductionto the subject. For example, antibody production may be potentiated ifthe peptides (and/or other peptidic modulatory molecule according to thepresent invention) are introduced with an adjuvant. Concentration of thepeptide or other molecule may also be important. In any case, one ofordinary skill in the art could easily determine conditions for antibodyproduction, as opposed to having the effect potentiated or mediateddirectly through the peptides and/or other peptidic modulatory moleculesaccording to the present invention.

The modulatory chemokine-binding peptide molecules of the presentinvention could therefore be useful for treating a disease selected fromthe group consisting of inflammation (primary or secondary e.g. uveitis,bowel inflammation), allergy, non-optimal immune response, autoimmunereaction (including rheumatoid arthritis, systemic lupus erythematosis,multiple sclerosis and others), delayed-type hypersensitivity, allograftrejection, diabetes, sepsis, cancer and any type of malignant cellgrowth, (including, but not limited to breast cancers such asinfiltrating duct carcinoma of the breast or other metastatic breastcancers, lung cancers such as small cell lung carcinoma, bone cancers,bladder cancers such as bladder carcinoma, rhabdomyosarcoma,angiosarcoma, adenocarcinoma of the colon, prostate or pancreas, orother metastatic prostate or colon cancers, squamous cell carcinoma ofthe cervix, ovarian cancer, malignant fibrous histiocytoma, skin cancerssuch as malignant melanoma, lymphomas and leukemia, leiomyosarcoma,astrocytoma, glioma and heptocellular carcinoma), acute and chronicbacterial and viral infections, vasculitis, arthritis, colitis,psoriasis, atherosclerosis, Graves disease, anorexia nervosa;hemorrhagic shock caused by septicemia, HIV infection in AIDS,pemphigus, asthma, renal diseases, liver diseases, bone marrow failure,vitiligo, alopecia, and myositis, hypertension and reperfusion ischemia.

The examples provided below, describe certain experiments performed withthe peptidic modulatory of the present invention, demonstrating thebinding efficacy of these molecules to the various chemokines. It alsodemonstrates the efficacy of the peptidic chemokine-binding modulator ina biological system. In addition, the examples also describeformulations for administering the compounds of the present invention,and methods of treatment thereof.

EXAMPLE 1 Efficacy of the Peptides of the Present Invention

This Example demonstrates the efficacy of the peptidic chemokine-bindingmodulators of the present invention, in a number of different assays,including binding assays and assays for measuring a functionalbiological effect.

Materials and Methods

Chemokines

Recombinant chemokines were ordered from PeproTech, Inc. (Rocky Hill,N.J. USA). Human SDF-1α (Cat. No. 300-28A), human MIG (Cat. No. 300-26)and human IL-8, (72 amino acids) (Cat. No. 200-08M), belong to thealpha-chemokines (C-X-C) family. Human MCP-1 (MCAF) (Cat. No. 300-04)and human Eotaxin (Cat No. 300-21) belong to the beta-chemokines (C-Cfamily). All chemokines were prepared according to the companyrecommendations.

Peptide Synthesis

Peptides were synthesized in the Weizmann Institute of Science, Rehovot,Israel, in order to perform tests for characterization of theirinfluence on the biological activity of the chemokines. The format ofthe various synthesized peptides was as follows: The cyclic peptides,ACX₇CGGGSK-biotin-G and the linear peptides, X₁₂GGGSK-biotin-G. Thepeptides were biotinylated on their C-termini; the biotin will serve asa detector during the following experiments. Each synthetic peptide wasdissolved to concentration of 1 mg/ml (˜0.6 mM) in 4% DMSO (DimethylSulphoxide, Sigma, Cat. # D-2650).

ELISA Analysis of the Synthetic Chemokine-binding Peptide

NUNC-Immuno maxisorp plates (Cat. No. 4-42404) were coated with theappropriate chemokine (0.1 ml/well, 0.1-1.0 μg/ml in 0.1 M NaHCO₃, pH8.6), overnight at 4° C. The plates were then blocked with 0.2 ml/wellof blocking buffer (5 mg/ml BSA in 0.1 NaHCO₃). Control wells weretreated with blocking buffer alone, with no addition of target protein(chemokine). The plates were washed 6 times with PBST (0.1% TWEEN® 20(polyoxyethylene (20) sorbitan monolaurate) in PBS), followed byincubation for 45 minutes at room temperature with 10-fold serialdilutions of individual synthetic peptides (10 pg-10 μg) with 1%BSA(PBST-BSA)/well. After the plates were washed 6 times with PBST, thebound peptides were probed by HRP-SA Conjugate, diluted 1:10,000 to1:20,000 in PBST-BSA, 0.1 ml/well for 45 minutes at room temperature.The target-bound synthetic peptides probed with HRP-SA were quantifiedby DAKO TMB one-step substrate system, followed by the addition of stopsolution, HCl—H₂SO₄ mixture (0.1 ml/well) (1N HCl, 3N H₂SO₄). Theresults were analyzed by ELISA reader at OD₄₅₀.

T-Cell Purification from Fresh Blood

50 ml blood was added to 10 ml Dextran (Dextran T-500 6% w/v) in PBS(phosphate buffer saline), and 7 ml citrate buffer (25 g citrate, 8 gcitric acid in 500 ml PBS). The solution was incubated for 30 min at 25°C. 10 ml Ficoll 1077 (Sigma) was added to the bottom of the tube. Thetube was then centrifuged at 2,000 rpm for 30 min, at 18° C., (with thebrake mode of the centrifuge off). The interphase was collected andwashed twice with 8 ml PBS-50% FCS (fetal calf serum), followed bycentrifugation at 1,400 rpm, for 5 min, at 18° C. The cells werere-suspended in PBS-5% FCS at a concentration less then 10⁸/ml. 2 ml ofthe cell solution were applied and incubated for 45 min at 25° C. on aPerspex Nylon wool column, which was pre-soaked with PBS-5% FCS. Eachcolumn was washed with 8 ml PBS-5% FCS and the cells (T-cells anderythrocytes) were eluted by 50 ml of 5 mM EDTA in PBS. A red pellet wasobtained by centrifugation at 1,400 rpm, at 4° C., for 5 min, with thebrake on. In order to perform lysis of the erythrocytes, the red pelletwas re-suspended in 5 ml lysis-buffer (155 mM NH₄Cl, 10 mM KHCO₃, 0.1 mMEDTA, X0.1PBS) for 4 min, followed by immediate addition of 50 ml ofPBS-EDTA.

Following centrifugation at 1,400 rpm, at 4° C., for Smin with brake on,the pellet was washed again with 50 ml PBS-EDTA and re-centrifuged underthe same conditions. A white pellet was obtained and re-suspended inRPMI/10% FCS/L-glutamine/sodium pyruvate/antibiotics at a concentrationof 3^(x)10⁶ cells/ml. The cells were incubated for 2 h at 37° C.,followed by collection of the non-adherent cells. The cells were readyfor use in experiments after overnight incubation at 37° C.

Preparation of Adhesive Substrates

Human VCAM-1 (1 μg/ml) and SDF-1α (intact or heat-inactivated) (2 μg/ml)were dissolved in PBS buffered with 20 mM bicarbonate, pH 8.5, andincubated on a polystyrene plates overnight at 4° C. The plates werethen washed three times and blocked with human serum albumin (20 mg/mlin PBS) for 2 h at 37° C.

Biovalidation

Laminar flow assays were performed as follows. Polystyrene plates (B.D)were coated with soluble VCAM-1 at 10 μg/ml in the presence of 2 μg/mlHSA carrier. The plates were washed three times with PBS and blockedwith HSA (20 μg/ml in PBS) for 2 hrs at room temperature. Alternatively,washed plates were coated with 10 μg/ml MIG chemokine in PBS for 30 minat room temperature, before being blocked with HSA. The plates wereassembled as the lower wall of a parallel wall flow chamber and mountedon the stage of an inverted microscope. The peptide, as describedpreviously, (10 μg/ml) was allowed to settle on the substrate coatedchamber wall for 10 min, at 37° C. and then washed. T cells (5×10⁶/ml,purity >98%) were suspended in binding buffer, perfused into the chamberand allowed to settle on the substrate coated chamber wall for 1 min, at37° C. Flow was initiated and increased in 2 to 2.5 fold incrementsevery 5 sec. generating controlled shear stresses on the wall. Cellswere visualized in a 20x objective of Nikon DIAPHOT TMD BiologicalInverted Microscope (Nikon, Japan) and photographed with a longintegration LIS-700 CCD video camera (Applitech; Holon, Israel),connected to a video recorder (AG-6730 S-VHS, Panasonic, Japan). Thenumber of adherent cells resisting detachment by the elevated shearforces was determined after each interval by analysis of videotaped cellimages, and was expressed as the percent of originally settled cells.All adhesion experiments were performed at least three times on multipletest fields.

Results

Identification of Chemokine Binding Peptides with Antagonistic Effect

The present invention identifies chemokine-binding peptides withbiological activity. The binding specificities of the various peptidesto the chemokines were determined by screening against BSA, Actin andFibronectin-coated wells as negative controls in parallel experiments.The binding level of the peptides to the various chemokines, whichreached at least two fold of the binding level to the control proteins,was considered a specific binding. The different peptides that werefound to bind to chemokines, are listed in Table 1.

Graphical representations of four examples of such peptide-containingcarriers, from the list in Table 1, are shown in FIG. 1. As can be seen,each of those peptides was found to bind to at least one differentchemokine, which was chosen to be presented in this graph: peptideBKT-P1 was found to bind SDF-1α; peptide BKT-P3 was found to bind, inthis representation, to MCP-1; peptide BKT-P22, was found to bindEotaxin and peptide BKT-P37, was found to bind IL-8. The specificity ofthe binding was calculated by comparing the binding level of theproteins to chemokines to that of binding level to control proteins, asexplained above. It should also be noted that the binding of each of thepeptides shown in FIG. 1 is not necessarily the only binding capabilityshown by those peptides.

Peptides that showed affinity/binding to one or more chemokines werethen analyzed in several individual experiments, and were chosen forfurther analysis. The specific binding of the peptides was detected byscreening methodology, using ELISA, as described in “Materials andMethods”, employing microplates coated with the various chemokines to bechecked. The binding specificities of the various peptides to thevarious chemokines were determined by screening against BSA, Actin andFibronectin-coated wells as negative controls in parallel experiments.

As can be seen in FIG. 2, peptides that can specifically bind to onespecific chemokine, such as BKT-P9, were identified (FIG. 2A). Inaddition, peptides that can bind, specifically to more than onechemokine, such as BKT-P1, were also identified (FIG. 2B). Again, thespecificity of the binding, either to one chemokine or more, wasestablished by comparing binding level with the control binding level tonon-related proteins, (shown in the Figure as a broken line). Controlbinding showed a level of one fold increase. As such, each of thepeptides that showed a level of binding to a particular chemokine ofless than, or close to, such a one-fold increase, was considered to benon-specifically bound to that chemokine. Specific binding wasconsidered as that which showed at least a two fold increase over thecontrol binding level. Peptides that specifically bound to one or morechemokines were chosen for further examination and for analysis with abiovalidation assay, to prove their ability to not only bind the variouschemokines but also to modulate the biological activity of thosechemokines.

According to the present invention, two families of chemokine-bindingpeptides were identified. These families contain peptides with similaramino acid compositions, a high percentage of histidines, and are alsocharacterized by overall positive electrostatic charge.

A detailed list of the potential consensus sequences of the two familiesis presented in Tables 2 and 3. The peptides of family no. 1 (Table 2)bind to MIG, MCP-1, IL-8, SDF-1α and Eotaxin, as illustrated in Table 2,and are predominantly composed of the amino acids H, S, A, L, I, K, R, Tand P. Each peptide in this family contains at least 2 histidinesdistributed along the molecule. The abundance of positively chargedamino acids such as H, K and R, results in peptides having an overallpositive charge. The remaining amino acids, mentioned above, mightparticipate in the determination of the three dimensional structure ofthe peptides.

Table 3 shows a family of peptides (family no. 2) that bind mostly toMCP-1 and in individual cases to IL-8, SDF-1α and Eotaxin (illustratedin the Table). The binding motif for peptides in this family ispredominantly composed of the amino acids H, P, T, L, R, W, F, whileeach peptide also features at least two histidines, one next to theother. The abundance of positively charged amino acids such as H and R,resulted in peptides having an overall positive charge; The remainingamino acids, mentioned above, may participate in the determination ofthe three dimensional structure of the peptides.

As defined here, a consensus sequence is composed of an amino acidsequence that is found repeatedly in a group of peptides that bindvarious chemokines and probably have certain biological functions incommon. The group or family of such peptides is characterized by theconsensus sequence, high abundance of the amino acid histidine andoverall positive electrostatic charge. These peptides may be describedas potential agonists or antagonists of chemokines.

The binding of one synthetic peptide, BKT-P3 (family 1), to variouschemokines is shown in FIG. 3. As can be seen, this peptide binds to allfive chemokines tested, in contrast to the control peptide, BKT-P18,which does not belong to the family, and also shows no binding to thechemokines. As discussed above, the level of binding was calculated bycomparing binding of the peptides to the chemokines with peptide bindingto controls. Level of control binding was defined as level 1 (up to aone-fold increase in binding). This control binding level is illustratedin the Figure as a dashed line. It can be seen that although BKT-P3binds several different chemokines, the level of the binding isdifferent in each case, which suggests different affinities and maybedifferent influences on the activity of each of the chemokines.

Further examination of BKT-P3 for efficacy in biovalidation wasperformed to check the ability of BKT-P3 to bind and modulate theactivity of MIG chemokine, to which BKT-P3 showed the highest bindinglevel (FIG. 3). The result of the experiment is graphically illustratedin FIG. 4.

As can be seen in FIG. 4B, when a control peptide (which does not belongto family 1 and which also does not bind to the chemokine being examined(MIG)) was added to the flow chamber in order to test for its ability tobind to and modulate the activity of MIG, no influence on the activityof MIG was seen. MIG activity continued to show the same level ofactivity (about 25%-30% of arrested cells) as was seen in the absence ofpeptide. Hence, as can be seen in FIG. 4B, the same percentage ofarrested cells could be detected in the presence of the chemokine, withor without the addition of BKT-P10 (the control peptide) (FIG. 4B,Mig+p10 and Mig, respectively). On the other hand, when BKT-P3, whichwas previously found to bind MIG chemokine, was added to the flowchamber, the percentage of arrested cells that reached about 25-30% inthe presence of Mig (FIG. 4A, Mig), was dramatically reduced (FIG. 4A,Mig+p3) to the control level, achieved in the presence of VCAM-1 alone,with no addition of chemokine (FIG. 4A, control). These results revealedan obvious antagonistic effect of BKT-P3 against human MIG, thechemokine to which it was able to bind (FIG. 4A), in contrast to thenon-binding control peptide, BKT-P10 (FIG. 4B).

Table 4 shows a summary of the biological activity results for fourrepresentative synthetic peptides belonging to family 1. Variousdifferent chemokines were used in the biovalidation assay together withthe adhesion receptor VCAM-1, in order to activate the adhesion of Tcells in the system (as described in “Materials and Methods”). As can beseen in Table 4, all the peptides that were checked clearly showedantagonistic effect on the various chemokines that were introduced intothe flow chamber. On the other hand, although all the peptides showedantagonistic effects, the efficiency of the effect varied between thedifferent peptides and between the same peptides tested againstdifferent chemokines. Thus, BKT-P3 caused complete arrest of thebiological activity of both chemokines that were checked, MIG and IL-8.BKT-P2, which shows high sequence similarity to BKT-P3, caused onlyabout 50% reduction in the activity of MIG. BKT-P45, which caused 100%abolishment of the activity of IL-8, as did BKT-P3, only reduced theactivity of MIG by about 20%. BKT-P39, on the other hand, which had noeffect on IL-8, caused 100% blocking of the activity of Eotaxin. Ittherefore seems that although the sequence similarity between themembers of the family is quite high, and causes similar biologicalactivity (antagonistic activity), the particular order of specificsignificant residues within the peptide sequence is also important.

Table 5 shows a summary of the biological activity results for tworepresentative synthetic peptides listed in Table 1, neither of whichbelong to the previously described families (family 1 and family 2). Asdescribed in the previous experiment, various different chemokines wereused in the biovalidation assay, together with the adhesion receptorVCAM-1, in order to activate the adhesion of T cells in the system (asdescribed in “Materials and Methods”). As can be seen in Table 5, eachof the tested peptides showed entirely different behavior. While BKT-p23showed an antagonistic effect with two of the four chemokines that itwas allowed to bind, although at different efficiencies, BKT-P6 showedan obvious agonistic effect on SDF1α, while having no effect on MIGactivity. BKT-P23 blocks 100% of Eotaxin activity but only about 20% ofthe activity of SDF-1α, and has no effect on the activity of either IL-8or MIG. BKT-P6 enhances SDF-1α activity by at least twenty fold, and hasno effect on MIG activity. It therefore appears, that although there isonly slight sequence similarity between BKT-P23 and the members offamilies 1 and 2, the fact that this peptide carries an overall positivecharge, as do the members of families 1 and 2, probably contributes toits ability to act as an antagonist of various chemokines, therebysomewhat resembling the activity of the members of the two families(BKT-P39 for example). On the other hand, BKT-P6 acts as an agonist.This peptide is a circular peptide and differs in its sequencecomposition, compared to the two families. BKT-P6 is a highly polarmolecule, and this property, as well as its circular configuration andthe resulting 3D structure, may contribute to its activity.

It is also important to note that this peptide (BKT-P6) was the firstchemokine agonist to be identified. Such agonists could clearly beimportant for a number of different reasons. For example, a designedchemokine agonist, whether in peptide form and/or a peptide derivative(eg with one or more chemical bonds substituted and/or other moleculessubstituted for one or more amino acids), could be useful in combinationwith other chemokines, for synergistic treatment, and/or as a substitutefor a chemokine in a therapeutic situation in which the chemokine has adesired effect on the subject. For example, pre clinical studies haveshown that MCP-1 is a powerful stimulator of vessel formation. Thischemokine is in advanced stages of development for clinical use inrestenosis. Augmenting the activity of MCP-1 may lead to better outcomeof therapy.

As another example, HCV (herpes cytomegalovirus) infection is resolvedby the immune system in only 20% of cases, while the other 80% of casesdeveloped chronic disease which may lead to a requirement for livertransplantation and/or liver cancer, such as hepatocellular carcinoma.Augmenting the activity of chemokines such as Mig and IP-10, whichparticipate in the regulation of the immune response against HCV, mayalso lead to better outcome of therapy in this situation.

Further examination of BKT-P3 and BKT-P2 (both belonging to family 1)for the immunity of these peptides was performed, by injecting thosepeptides to mice. The result of the experiment is graphicallyillustrated in FIG. 5. The control, pre-immune serum of the same mice towhich the peptides were injected showed no binding to the two peptides(the O.D. results of both pre-immune sera were the same as the O.D.achieved by binding of all sera to the plastic with no peptideaddition). On the other hand, the immune sera showed high O.D. levels,in the presence of the two peptides. These results showed that the seracollected from the mice after injection of the peptides containantibodies against the two peptides.

The results summarized above are very important, since it is known thatone chemokine can bind to more than one receptor and as such, can beinvolved in more than one pathological disorder. On the other hand, morethen one chemokine can bind to one receptor, which means that severalchemokines might be involved in one pathological disorder. So, in somecases, in order to block one pathological disorder, it may be necessaryto block the activity induced by more than one chemokine. Alternatively,in some cases, the activity of only one specific chemokine may need tobe blocked in order to interfere with the progression of one specificpathological disorder.

The results, shown in FIGS. 2 and 3, and Tables 4 and 5, demonstratethat there is a possibility of modulating either the activity of onespecific chemokine, or the activity of-several different chemokines thatare involved in the development of one pathological disorder, andthereby interfering with the progression of the various diseases.

The results shown in FIG. 5 demonstrate the immunogenicity of thepeptides. The ability to raise antibodies against the peptides, whichwould therefore mimic the chemokine receptors, provides support to otherapplications of the present invention, including but not limited to, thedevelopment of vaccines against the chemokine receptors. These receptorsare known to be involved in various pathological disorders. Suchvaccines would thus enable treatment and/or prevention of suchdisorders.

EXAMPLE 2 Efficacy of the Peptides of the Present Invention

This Example demonstrates the efficacy of the peptidic chemokine-bindingmodulators of the present invention, in a number of different assays,including binding assays and assays for measuring a functionalbiological effect both in vitro and in vivo.

Materials and Methods

Chemokines

Recombinant chemokines were ordered from PeproTech, Inc. (Rocky Hill,N.J. USA). Human SDF-1α (Cat. No. 300-28A), human MIG (Cat. No. 300-26)and human IL-8, (72 amino acids) (Cat. No. 200-08M), belong to thealpha-chemokines (C-X-C) family. Human MCP-1 (MCAF) (Cat. No. 300-04)and human Eotaxin (Cat No. 300-21) belong to the beta-chemokines (C-Cfamily). All chemokines were prepared according to the companyrecommendations.

Peptide Synthesis

Synthetic peptides were ordered from BioSight Ltd, Karmiel, Israel, inorder to perform tests for characterization of their influence on thebiological activity of the chemokines. The format of the varioussynthesized peptides was as follows: The cyclic peptides,ACX₇CGGGSK-biotin-G and the linear peptides, X₁₂GGGSK-biotin-G. Thepeptides were biotinylated on their C-termini; the biotin serves as adetector during the following experiments. Each synthetic peptide isdissolved to concentration of 1 mg/ml (˜0.6 mM) in 4% DMSO (DimethylSulphoxide, Sigma, Cat. # D-2650).

ELISA Analysis of the Synthetic Chemokine-Binding Peptide

NUNC-Immuno maxisorp plates (Cat. No. 4-42404) are coated with theappropriate chemokine (0.1 ml/well, 0.1-1.0 μg/ml in 0.1 M NaHCO₃, pH8.6), overnight at 4° C. The plates are then blocked with 0.2 ml/well ofblocking buffer (5 mg/ml BSA in 0.1 NaHCO₃). Control wells are treatedwith blocking buffer alone, with no addition of target protein(chemokine). The plates are washed 6 times with PBST (0.1% TWEEN®20(polyoxyethylene (20) sorbitan monolaurate) in PBS), followed byincubation for 45 minutes at room temperature with 10-fold serialdilutions of individual synthetic peptides (10 pg-10μg) with 1%BSA(PBST-BSA)/well. After the plates are washed 6 times with PBST, thebound peptides are probed by HRP-SA Conjugate, diluted 1:10,000 to1:20,000 in PBST-BSA, 0.1 ml/well for 45 minutes at room temperature.The target-bound synthetic peptides probed with HRP-SA are quantified byDAKO TMB one-step substrate system, followed by the addition of stopsolution, HCl-H₂SO₄ mixture (0.1 ml/well) (1N HCl, 3N H₂SO₄). Theresults are analyzed by ELISA reader at OD₄₅₀.

T-Cell Purification from Fresh Blood

50 ml blood is added to 10 ml Dextran (Dextran T-500 6% m/v) in PBS(phosphate buffer saline), and 7 ml Citrate buffer (25 g citrate, 8 gcitric acid in 500 ml PBS). The solution is incubated for 30 min at 25°C. 10 ml Ficoll 1077 (sigma) is added to the bottom of the tube. Thetube is then centrifuged at 2,000 rpm for 30 min, at 18° C., (with thebrake mode of the centrifuge off). The interphase was collected andwashed twice with 8 ml PBS-5% FCS (fetal calf serum) followed bycentrifugation at 1,400 rpm, for 5 min, at 18° C. The cells arere-suspended in PBS-5% FCS at a concentration less then 10⁸/ml. 2 ml ofthe cells solution are applied and incubated for 45 min at 25° C. on aPerspex Nylon wool column, which is pre-soaked with PBS-5% FCS. Eachcolumn is washed with 8 ml PBS-5% FCS and the cells (T-cells anderythrocytes) are eluted by 50 ml of 5 mM EDTA in PBS. A red pellet isobtained by centrifugation at 1,400 rpm, at 4° C., for 5 min, with thebrake on. In order to perform lysis of the erythrocytes, the red pelletis re-suspended in 5 ml lysis-buffer (155 mM NH₄Cl, 10 mM KHCO₃, 0.1 mMEDTA, X0.1PBS) for 4 min, followed by immediate addition of 50 ml ofPBS-EDTA. Following centrifugation at 1,400 rpm, at 4° C., for 5 minwith brake on, the pellet is washed again with 50 ml PBS-EDTA andre-centrifuged under the same conditions. A white pellet is obtained andre-suspended in RPMI/10% FCS/L-glutamine/sodium pyruvate/antibiotics ata concentration of 3^(X)10⁶ cells/ml. The cells are incubated for 2 h at37° C., followed by collecting of the non-adherent cells. The cells areready for experiments after overnight incubation at 37° C.

Preparation of Adhesive Substrates

Human VCAM-1 (1 μg/ml) and SDF-1α (intact or heat-inactivated) (2 μg/ml)are dissolved in PBS buffered with 20 mM bicarbonate, pH 8.5, andincubated on a polystyrene plates overnight at 4° C. The plates are thenwashed three times and blocked with human serum albumin (20 mg/ml inPBS) for 2 h at 37° C.

Biovalidation

Laminar flow assays are performed as follows. Polystyrene plates (B.D)are coated with soluble VCAM-1 at 10 μg/ml in the presence of 2 μg/mlHSA carrier. The plates are washed three times with PBS and blocked withHSA (20 μg/ml in PBS) for 2 hrs at room temperature. Alternatively,washed plates are coated with 10 μg/ml MIG chemokine in PBS for 30 minat room temperature, before being blocked with HSA. The plates areassembled as the lower wall of a parallel wall flow chamber and mountedon the stage of an inverted microscope. The peptide, as describedpreviously, (10 μg/ml) is allowed to settle on the substrate coatedchamber wall for 10 min, at 37° C. and then washed. T cells (5×10⁶/ml,purity >98% ) are suspended in binding buffer, perfused into the chamberand allowed to settle on the substrate coated chamber wall for 1 min, at37° C. Flow is initiated and increased in 2 to 2.5 fold increments every5 sec. generating controlled shear stresses on the wall. Cells arevisualized in a 20x objective of Nikon DIAPHOT TMD Biological InvertedMicroscope (Nikon, Japan) and photographed with a long integrationLIS-700 CCD video camera (Applitech; Holon, Israel), connected to avideo recorder (AG-6730 S-VHS, Panasonic, Japan). The number of adherentcells resisting detachment by the elevated shear forces is determinedafter each interval by analysis of videotaped cell images, and isexpressed as the percent of originally settled cells. All adhesionexperiments are performed -at least three times on multiple test fields.

In-Vitro Migration Assay

The migration of purified T cells in vitro towards chemokines isdetermined by a trans-well migration. The cells are viewed in aninverted microscope, before starting the procedure. Followingcentrifugation of the cells at 1,300 rpm, for 5 min, at R.T., the cellsare re-suspended in RPMI/1% FCS for a concentration of 1.8^(×)10⁶ cells/ml. In parallel, the trans-wells (Costar 3421, Corning Costar,Cambridge, MA) are coated with 100μl of fibronetcin, 10μg/ml for 1 hr at37° C. Subsequently, 100 μl of the treated cells are added to the upperchamber of the trans-well, and 600 82 l of RPMI+1% FCS is added to thelower chamber of the transwell, with or without 100 ng/ml chemokine tobe checked, with or without various concentrations of the peptide to bechecked . The experiment is performed in triplicates. The incubationtakes place for 4 h or 5 h at 37° C. Following staining with Trypan-blueor Alamar +, the cells in the lower chamber of the trans-well arecounted, by FACS, cell counter or fluorometer, respectively. Thepercentage of migration is calculated as the percent of migrated cell(minus the background), from the total number of the cells that areloaded on the trans-well. Percentage of migration inhibition iscalculated as the percentage of migration in the presence of thepeptides compared to the migration of cells subjected to the sametreatment, in the absence of the peptides.

In Vivo Migration Assay

Wild-type (WT) male BALB/c mice (18-25 g), with age ranging between 8and 12 weeks, are used throughout these experiments. Animals are fedcommercial rodent chow and are housed in a temperature-controlled roomwith free access to water and food.

The migration of Eosinophils or Neutrophils in vivo to the peritoneum,in response to various chemokines, is determined by injection of thechemokine to be checked, with or without (control) the peptide inquestion, i.v. into the peritoneum of the mice. The animals aresacrificed at different times after the injection and their peritonealcavities are washed with 2-4 ml of PBS and the total cell counts aredetermined.

Results

Peptide Synthesis

Eight different peptides were purchased (Table 6). The peptides to beordered were determined as previously described. BKT-P3 was ordered as a12 amino acid peptide, without the linker (discussed in “Materials andMethods”). The peptide had previously been found to be effective in thisconfiguration, and will be used in both in vivo experiments and Absproduction. BKT-P2 and BKT-P39, which belong to family 1, were orderedfor further studies, since, in spite of their resemblance to BKT-P3,they are effective at promoting activities related to chemokines otherthan those for which BKT-P3 is effective. BKT-P49, which also belongs tofamily 1, was ordered as a cyclic representative of family 1. The restof the peptides that were ordered, are peptides that do not belong tofamily 1 and were ordered as such. The other two peptides, BKT-P35 and57 were chosen as representatives of linear and circular peptides thatdo not belong to the recognized families. The last peptide, BKT-P 18, isa control peptide, which showed no binding to the various chemokines andwas used as such. All the ordered peptides are checked for their abilityto bind the various chemokines and modulate their activity, both invitro and in vivo, as per the previously described experiments.

EXAMPLE 3 Methods and Compositions for Administration

The peptides of the present invention, and their homologues, derivativesor related compounds, hereinafter referred to as the “therapeutic agentsof the present invention”, can be administered to a subject by variousways, which are well known in the art. Hereinafter, the term“therapeutic agent” includes a peptidic chemokine-binding modulator, aspreviously defined, including but not limited to, any of the abovebiologically useful peptides and their homologs, analogs,peptidomimetics and derivatives thereof.

Hereinafter, the term “subject” refers to the human or lower animal towhich the therapeutic agent is administered For example, administrationmay be done topically (including ophthalmically, vaginally, rectally,intranasally and by inhalation), orally, or parenterally, for example byintravenous drip or intraperitoneal, subcutaneous, or intramuscularinjection.

Formulations for topical administration may be included but are notlimited to lotions, ointments, gels, creams, suppositories, drops,liquids, sprays and powders. Conventional pharmaceutical carriers,aqueous, powder or oily bases, thickeners and the like may be necessaryor desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, sachets,capsules or tablets. Thickeners, diluents, flavorings, dispersing aids,emulsifiers or binders may be desirable.

Formulations for parenteral administration may include but are notlimited to sterile aqueous solutions, which may also contain buffers,diluents and other suitable additives.

Dosing is dependent on the severity of the symptoms and on theresponsiveness of the subject to the therapeutic agent. Persons ofordinary skill in the art can easily determine optimum dosages, dosingmethodologies and repetition rates.

EXAMPLE 4 Methods of Treatment with the Compounds

As noted above, the therapeutic agents of the present invention havebeen shown to be effective modulators of cell adhesion and cellmigration that characterize inflammatory reaction, cancer metastasis andany other suitable conditions in which a particular target ligand bindsto its target receptor. The following example is an illustration only ofa method of treating an inflammatory condition, cancer metastasis andany other suitable condition involving cell migration, with thetherapeutic agent of the present invention, and is not intended to belimiting.

The method includes the step of administering a therapeutic agent, in apharmaceutically acceptable carrier, to a subject to be treated. Thetherapeutic agent is administered according to an effective dosingmethodology, preferably until a predefined endpoint is reached, such asthe absence of a symptom of the inflammatory condition, blockage oftumor metastasis and any other suitable condition in the subject, or theprevention of the appearance of such a condition or symptom in thesubject.

The modulatory chemokine-binding peptide molecules of the presentinvention could therefore be useful for treating a disease selected fromthe group consisting of inflammation (primary or secondary e.g. uveitis,bowel inflammation), allergy, non-optimal immune response, autoimmunereaction (including rheumatoid arthritis, systemic lupus erythematosis,multiple sclerosis and others), delayed-type hypersensitivity, allograftrejection, diabetes, sepsis, cancer and any type of malignant cellgrowth, (including, but not limited to breast cancers such asinfiltrating duct carcinoma of the breast or other metastatic breastcancers, lung cancers such as small cell lung carcinoma, bone cancers,bladder cancers such as bladder carcinoma, rhabdomyosarcoma,angiosarcoma, adenocarcinoma of the colon, prostate or pancreas, orother metastatic prostate or colon cancers, squamous cell carcinoma ofthe cervix, ovarian cancer, malignant fibrous histiocytoma, skin cancerssuch as malignant melanoma, lymphomas and leukemia, leiomyosarcoma,astrocytoma, glioma and heptocellular carcinoma), acute and chronicbacterial and viral infections, vasculitis, arthritis, colitis,psoriasis, atherosclerosis, Graves disease, anorexia nervosa;hemorrhagic shock caused by septicemia, HIV infection, pemphigus,asthma, renal diseases, liver diseases, bone marrow failure, vitiligo,alopecia, and myositis, hypertension and reperfusion ischemia.

EXAMPLE 5 Method of Immunization with the Peptides

This Example provides a non-limiting illustrative method according tothe present invention for inducing an immune response in a subject withone or more of the peptides (or peptidomimetics, homologs, derivativesetc as previously described) according to the present invention. Thismethod would preferably enable blockage of binding of a chemokine to itsreceptor, and/or immune sequestration of the receptor, by causing theimmune system of the subject to produce an antibody against thereceptor. Since the peptides according to the present invention bindchemokines, an antibody against such a peptide would be expected to bindto the receptor of the chemokine and/or to block binding of thechemokine to the receptor. In any case, such an immunization would beexpected to block or at least reduce the biological activity induced bythe receptor.

Examples of such methods with regard to another protein, the heparanaseenzyme, are described in PCT Application No. WO 03/006645, herebyincorporated by reference as if fully described herein.

Briefly, the chemokine biological activity is inhibited by elicitationof an immune response to the peptide according to the present inventionfollowing administration of an effective amount of the peptide. In thecontext of the present invention, the peptide is administered in anamount effective to elicit an immune response, including a humoral orcell-mediated immune response, against the native receptor. The immuneresponse is preferably an active immunity that inhibits, that is,prevents, slows, or stops, the chemokine-induced biological activity.Therefore, in the context of the present inventive methods, suchbiological activity need not be completely abrogated. It should beappreciated that the immune response against the receptor can beelicited either directly or indirectly.

In an alternative embodiment, the peptide of the present invention canbe modified in various ways known to one of skill in the art, forexample, by co-administering with or conjugating or genetically fusingit to an immunogenic reagent. Conjugation or fusion to an immunogenicreagent can stimulate an immune response or augment the existing immuneresponse elicited by the peptide. These conjugates and fused moleculescan be prepared by any of the known methods for coupling or fusingantigens to carriers or fusion molecules. The conjugates can also beprepared recombinantly as fusion polypeptides by methods well known inthe art. The preferred method of conjugation is covalent coupling,whereby the antigen is bound directly to the immunogenic reagent.Moreover, coadministration can be such that the immunogenic reagent isadministered prior to, concurrently with, or subsequent to the peptide.

While the invention has been described with respect to a limited numberof embodiments, it will be appreciated that many variations,modifications and other applications of the invention may be made.

TABLE 1 Seq Peptide Name id no: Sequence of peptide BKT-P50 1 CAHLSPHKCBKT-P10 2 CDIPWRNEC BKT-P17 3 CDPLRQHSC BKT-P58 4 CDSLGHWLC BKT-P15 5CDYTTRHSC BKT-P59 6 CHGTLNPEC BKT-P56 7 CHHNLSWEC BKT-P60 8 CHIWTLASCBKT-P61 9 CHNTFSPRC BKT-P62 10 CIPLHASLC BKT-P63 11 CITTTSLSC BKT-P64 12CKLTTCKDC BKT-P65 13 CKNHTTFWC BKT-P66 14 CLKLLSRSC BKT-P67 15 CLLKAHPSCBKT-P68 16 CLNQLKQAC BKT-P69 17 CMNFPSPHC BKT-P70 18 CPQSPTYTC BKT-P5719 CPSSAIHTC BKT-P71 20 CPTSTARIC BKT-P72 21 CQASSFPSC BKT-P73 22CQPYFWYRC BKT-P14 23 CQTLTPSIC BKT-P74 24 CSKLGHLWC BKT-P75 25 CSKTPERIXBKT-P76 26 CSNNNRMTC BKT-P77 27 CSPILSLSC BKT-P16 28 CSPTNFTRC BKT-P7829 CSRPAMNVC BKT-P79 30 CSTKAYPNC BKT-P80 31 CSTSSCGSC BKT-P81 32CSYWGHRDC BKT-P13 33 CTAHDANAC BKT-P82 34 CTANSEKTC BKT-P83 35 CTHPKASMCBKT-P84 36 CTKTINGKC BKT-P85 37 CTNMQSPLC BKT-P86 38 CTPFTKLPC BKT-P8739 CTPTTDSIC BKT-P88 40 CTQQNGHPC BKT-P12 41 ACTTPSKHQC BKT-P89 42CTYNVAKPC BKT-P90 43 ACAPLMFSQC BKT-P48 44 ACHASLKHRC BKT-P91 45AHFSPNLLLGG BKT-P44 46 AHSLKSITNHGL BKT-P92 47 AKTLMPSPFPRT BKT-P93 48ASAVGSLSIRWQ/L/G BKT-P94 49 ASWVDSRQPSAA BKT-P95 50 CPQLTVGQHRT BKT-P851 DLPPTLHTTGSP BKT-P96 52 DSSNPIFWRPSS BKT-P97 53 EFLGVPASLVNP BKT-P5154 ESDLTHALHWLG BKT-P98 55 EVHSTDRYRSIP BKT-P99 56 FGLQPTGDIARR BKT-P957 FSMDDPERVRSP BKT-P100 58 FSPLHTSTYRPS BKT-P27 59 GDFNSGHHTTTR BKT-P2860 GPSNNLPWSNTP BKT-P33 61 GVHKHFYSRWLG BKT-P101 62 HAPLTRSPAPNLBKT-P102 63 HGSLTTLF/LRYEP BKT-P45 64 HHFHLPKLRPPV BKT-P55 65HHTWDTRIWQAF BKT-P54 66 HPTTPFIHMPNF BKT-P103 67 HRDPXS(P)PSAA/GRPBKT-P104 68 HNVTTRTQRLMP BKT-P49 69 HSACHASLKHRC BKT-P105 70HSACKLTTCKDG BKT-P6 71 HSACLSTKTNIC BKT-P106 72 IAHVPETRLAQM BKT-P107 73IFSMGTALARPL BKT-P108 74 INKHPQQVSTLL BKT-P7 75 ISPSHSQAQADL BKT-P46 76LDYPIPQTVLHH BKT-21 77 LFAAVPSTQFFR BKT-P22/38 78 LGFDPTSTRFYT BKT-P3779 LLADTTHHRPWP BKT-P109 80 LPWAPNLPDSTA BKT-P110 81 LQPSQPQRFAPTBKT-P111 82 LSPPMQLQPTYS BKT-P112 83 MHNVSDSNDSAI BKT-P113 84NSSMLGMLPSSF BKT-P114 85 NTSSSQGTQRLG BKT-P42 86 PGQWPSSLTLYK BKT-P23 87QIPQMRILHPYG BKT-P24 88 QIQKPPRTPPSL BKT-P115 89 QLTQTMWKDTTL BKT-P11690 QNLPPERYSEAT BKT-P117 91 QSLSFAGPPAWQ BKT-P118 92 QTTMTPLWPSFSBKT-P119 93 RCMSEVISFNCP BKT-P120 94 RSPYYNKWSSKF BKT-P39 95SAGHIHEAHRPL BKT-P40 96 SAISDHRAHRSH BKT-P121 97 SEPTYWRPNMSG BKT-P32 98SFAPDIKYPVPS BKT-P31 99 SFWHHHSPRSPL BKT-P3 100 SIFAHQTPTHKN BKT-P2 101SIPSHSIHSAKA BKT-P122 102 SIRTSMNPPNLL BKT-P123 103 SLPHYIDNPFRQ BKT-P29104 SLSKANILHLYG BKT-P124 105 SLVTADASFTPS BKT-P125 106 SMVYGNRLPSALBKT-P126 107 SPSLMARSSPYW BKT-P127 108 SPNLPWSKLSAY BKT-P1 109SQTLPYSNAPSP BKT-P128 110 SSTQAHPFAPQL BKT-P129 111 STPNSYSLPQAR BKT-P4112 STVVMQPPPRPA BKT-P34 113 SVQTRPLFHSHF BKT-P130 114 SVSVGMKPSPRPBKT-P131 115 SYIDSMVPSTQT BKT-P132 116 SYKTTDSDTSPL BKT-P133 117TAAASNLRAVPP BKT-P5 118 TAPLSHPPRPGA BKT-P134 119 TGLLPNSSGAGI BKT-P135120 TGPPSRQPAPLH BKT-P30 121 TLSNGHRYLELL BKT-P25 122 TPSPKLLQVFQABKT-P136 123 TPSTGLGMSPAV BKT-P137 124 TPVYSLKLGPWP BKT-P47 125TRLVPSRYYHHP BKT-P138 126 TSPIPQMRTVPP BKT-P139 127 TTNSSMTMQLQRBKT-P140 128 TTTLPVQPTLRN BKT-P141 129 TTTWTTTARWPL BKT-P142 130TVAQMPPHWQLT BKT-P143 131 TWNSNSTQYGNR BKT-P144 132 TWTLPAMHPRPA BKT-P26133 VHTSLLQKHPLP BKT-P35 134 VLPNIYMTLSA BKT-P145 135 VMDFASPAHVLPBKT-P146 136 VNQEYWFFPRRP BKT-P147 137 VYSSPLSQLPR BKT-P148 138VPPIS(R)TFLF(L)ST(K)S BKT-P149 139 VPPLHPALSRXN BKT-P43 140 VSPFLSPTPLLFBKT-P150 141 VSRLGTPSMHPS BKT-P151 142 WPFNHFPWWNVP BKT-P52 143WSAHIVPYSHKP BKT-P152 144 WWPNSLNWVPRP BKT-P53 145 YATQHNWRLKHE BKT-P153146 YCPMRLCTDC BKT-P154 149 YGKGFSPYFHVT BKT-P155 148 YPHYSLPGSSTLBKT-P156 149 YPSLLKMQPQFS BKT-P157 150 YQPRPFVTTSPM BKT-P158 151YSAPLARSNVVM BKT-P36 152 YTRLSHNPYTLS BKT-P41 153 YTTHVLPFAPSS BKT-P159154 YTWQTIREQYEM BKT-P6 155 CLSTKTNIC BKT-P6 156 ACLSTKTNIC BKT-P11 157CTTPSKHQC

TABLE 2 Bound Peptide Seq id chemokines name no: Peptide sequence (atleast) BKT-P3 100 SIFAHQTPTHKN MIG, IL-8, MCP-1 BKT-P2 101 SIPSHSIHSAKAMCP-1, Eotaxin, IL-8 BKT-P40 96 SAISDHRAHRSH IL-8 BKT-P39 95SAGHIHEAHRPL Eotaxin, SDF- 1alpha, IL-8 BKT-P48 44 ACHASLKHRC MCP-1BKT-P44 46 AHSLKSITNHGL MCP-1 BKT-P51 54 ESDLTHALHWL MCP-1 BKT-P49 69HSACHASLKHR MCP-1 BKT-P52 143 WSAHIVPYSHKP MCP-1 BKT-P53 145YATOHNWRLKHE MCP-1 BKT-P50 1 CAHLSPHKC MIG BKT-P33 61 GVHKHFYSRWLGEotaxin BKT-P54 66 HPTTPFIHMPNF MIG BKT-P34 113 SVQTRPLFHSHF EotaxinBKT-P26 133 VRTSLLQKHPLP MCP-1 Amino Acid Composition H = 33 S = 22 A =16 L = 15 I = 7 K = 10 R = 8 T = 9 P = 12 N = 3 G = 3 W = 4 Y = 3 V = 3E = 1 Q = 3 O = 1 D = 2

TABLE 3 Bound Peptide Seq id chemokines name no: Peptide sequence (atleast) BKT-P27 59 GDFNSGHHTTTR MCP-1 BKT-P45 64 HHFHLPKLRPPV IL-8, MCP-1, MIG BKT-P55 65 HHTWDTRIWQAF MCP-1 BKT-P46 76 LDYPIPQTVLHH MCP-1BKT-P37 79 LLADTTHHRPWP IL-8 BKT-P47 125 TRLVPSRYYHHP MCP-1 BKT-P56 7CHHNLSWEC SDF-1alpha BKT-P31 99 SFWHHHSPRSSPL Eotaxin Amino AcidComposition H = 18 P = 11 T = 9 L = 9 R = 6 W = 5 F = 4 D = 4 G = 2 N =1 S = 7 K = 1 V = 2 I = 2 Q = 1 Y = 2 A = 2

TABLE 4 Families 1 and 2 Biovalidation Results Level of Peptide Seq idPeptide Chemokine Antagonistic name no: Sequence checked Effect BKT-P3100 SIFAHQTPTHKN MIG +++ IL-8 +++ BKT-P2 101 SIPSHSIHSAKA MIG ++ BKT-P4564 HHFHLPKLRPPV IL-8 +++ MIG + BKT-P39 95 SAGHIHEAHRPL Eotaxin +++ IL-8− Legend: The percentage of antagonistic effect is as follows: + 20% ++50% +++ 100% − No effect

TABLE 5 Non-family members Biovalidation Results Seq Level Peptide idPeptide Chemokine of Kind of name no: Sequence checked Effect EffectBKT-P23 87 QIPQMRILHPYG EOTAXIN +++ Antagonist IL-8 − SDF-1α +Antagonist BKT-P6 71 HSACLSTKTNIC MIG − SDF-1α +++ Agonist Legend: Thepercentage of modulatory effect is as follows: + 20% ++ 50% +++ 100% −No effect

TABLE 6 Synthetic peptides ordered Seq id Chemokines Peptide name no:Peptide Sequence checked BKT-P3 100 SIFAHQTPTHKN MIG IL-8 BKT-P2 101SIPSHSIHSAKA MCP-1 EOTAXIN IL-8 BKT-P39 95 SAGHIHEAHRPL Eotaxin IL-8BKT-P49 69 HSACHASLKHRC — BKT-P6 71 HSACLSTKTNIC MIG SDF-1α BKT-P35 134VLPNIYMTLSA — BKT-P57 19 CPSSAIHTC — BKT-P18 — CKESATYFC —

REFERENCES

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Ma, Q., Jones, D., Borghesani, P. R., Segal, R. A., Nagasawa, T.,Kishimoto, T., Bronson, R. T. and T. A. Springer. 1998. Proc. Natl.Acad. Sci. USA, 95, 9448-9453.

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1. A method of inhibiting a binding of a chemokine to a chemokinereceptor, wherein said chemokine is selected from the group consistingof MCP-1 (monocyte chemotactic protein-1) and MIG (monokine induced bygamma interferon), the method comprising contacting the chemokine withan effective amount of a peptide comprising the amino acid sequence asset forth in SEQ ID NO:76.
 2. The method of claim 1, wherein saidpeptide binds to at least one of said chemokines and directly inhibits abiological activity of said chemokine by inhibition of binding of saidchemokine to said chemokine receptor.
 3. A method of inhibiting abinding of a chemokine to a chemokine receptor, wherein said chemokineis selected from the group consisting of IL-8 (interleukin-8) and MIG,the method comprising contacting the chemokine with an effective amountof a peptide comprising the amino acid sequence as set forth in SEQ IDNO:64.
 4. The method of claim 3, wherein said peptide binds to at leastone of said chemokines and directly inhibits a biological activity ofsaid chemokine by inhibition of binding of said chemokine to saidchemokine receptor.